PREGNANCY ASSOCIATED

BIOMOLECULES
Jerome A,SK Singh & SK Agarwal

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PREGNANCY

CONCEPTUS

MATERNAL SYSTEM

(Spencer et al.,2004)

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IFN- CG PL
CONCEPTUS

PAG EPF

ES

(Aschheim & Zondek,1927;Kappes et al.,1990; Bartol et al.,1985; Morton et al., 1987; Zoli et al.,1991; Henderson et al., 1993)
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These signals-are potential markers of pregnancy and embryonic viability as they influence pregnancy recognition and successful implantation. Conceptus derived substances are considered to be more precise and reliable markers of pregnancy and fetal well being. Conceptus secretes specific substances for its recognition by mother.
(Sousa et al.,2006;Szafranska et al.,2006)
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INTERFERON
Secretory product of the conceptus, from day 10 to 24 of gestation & peaks at day 17 in cattle and day 16 in sheep. (Bartol et al.,1985; Roberts et al.,2007) Prevents luteolysis by suppressing PGF2 secretion.

Prevents recurrence of estrus and ensures pregnancy to continue.

Up-regulates expression of no. of proteins and genes. Provide suitable environment locally to the conceptus. (c) Jerome A

Conti
Possessing a molecular weight of 20-24 kDa with 172 amino acids & considered as prime signal for MRP. (Knickerbockers et al.,1986) Remains within the uterine lumen and does not appear in measurable quantities in maternal blood or other body fluids. Therefore, can not be used as a marker for EPD.
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EARLY PREGNANCY FACTOR

Earliest known signal claimed to be specific for fertilization. First described in mice and later on to sheep,cattle and humans. (Mortan et al.,1974;Nancarrow et al.,1981) Characterized as glycoprotein or polypeptide with a potent immunosuppressive properties. (Noonan et al.,1979) Detected by Rosette inhibition test. (Morton et al.,1984) Serum RIT values low on the day of AI, increased on day 3 and remained high until embryo recovery on day 7 and subsequently decreased to a lower level by a week after embryo recovery. (Sakonju et al.,1993)
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Conti RIT assay is difficult, tedious, cumbersome and time consuming test. Indirect test, substances that have similar effects may confound the results. Therefore, it could not become popular test for routine pregnancy diagnosis in farm animals. Attempt to isolate a protein responsible for EPF activity has produced variable results without any convincing identification or characterization. (Mehta et al., 1989; Klima et al., 1992; Threlfall, 1994)
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Conti
Many diagnostic kits KEMS BioTest Ltd. (Littleton, CO), Early Conception Factor (ECF) test (Concepto Diagnostics, Knoxville, TN) & ECF dipstick test (Adams and Jordan, 1999; Concepto Diagnostics, 1999; Des coteaux et al.,2000) Detects pregnancy within 48 hrs after conception. More false-positive & false-negative results. So ECF test in present form is not a reliable method for determining early pregnancy. Cordoba et al. (2001) (c) Jerome A

ESTRONE SULPHATE
Major estrogen secreted from binucleate cells. Estimated in milk, plasma and urine.

(Henderson et al.,1993)
Mean conc. in buffalo varies between 14.8 to 23.6 ng/ml from second to ninth month of

pregnancy.

(Hung and Prakash,1990)

RIA/EIA was used to detect estrone sulphate. (Henderson et al.,1993;Prakash & Madan,1993)

Detects pregnancy at 31/2-4 months of

gestation i.e. too late to be considered as an early indicator of pregnancy.
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Chorionic Gonadotropins
In 1927, Aschheim and Zondek first reported the presence of a gonadotropic factor in the urine of pregnant women.

Known as human chorionic gonadotropin (hCG), glycoprotein secreted by the trophoblast.

Based on this, an accurate method of EPD in human was developed.

hCG is first detectable 8 to 11 days after conception.

In 1930, Cole and Hart demonstrated PMSG, now known as equine chorionic gonadotropin (eCG) produced from the endometrial cups composed of trophoblastic cells. Detectable from day 35-40 days of pregnancy. Such substance does not exist in ruminants, cannot be used as indicator for EPD. (c) Jerome A

PLACENTAL LACTOGEN
Synthesized by the binucleate cells & secreted throughout gestation. (Kappes et al., 1990) MW bovine- 32-34, ovine- 22 & caprine- 23 kDa, (Byatt et al., 1986) Mammogenesis, regulation of fetal growth, ovarian steroidogenesis & maternal inter-mediary metabolism. (Byatt et al., 1992) Concentration remains low & varies from 1 to 2 ng/ ml throughout gestation. (Beckers et al., 1982, Byatt et al.,1987) Since it is detected only from 3-4 month of gestation in cattle,it is not used as an indicator of early pregnancy. (Byatt, 1987)
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PREGNANCY ASSOCIATED GLYCOPROTEINS

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PREGNANCY ASSOCIATED GLYCOPROTEINS

Acidic Glycoproteins belonging to Aspartic Proteinase family. Molecular structure has more than 50 % AA sequence identity with Pepsin, Cathepsin D & E.
(Xie et al.,1991,1994)

Secreted by binucleate trophoblastic cells of placenta.

Constitute a group of pregnancyspecific proteins. Nearly 22 different PAGs in ruminants has been identified at different stage of gestation.
(Xie et al.,1991,1994,1997;Green et al., 2000; Garbayo et al., 2005) (c) Jerome A

PAG1
The first member of the family was isolated by Butler et al. (1982) from bovine placenta and was named as pregnancy specific protein B (PSPB). Subsequently, related proteins have been identified with the different names such as PAG1 (Xie et al., 1991; Zoli et al., 1991), PSP-60 (Mialon et al., 1993), SBU3 (Atkinson et al., 1993) in ruminant ungulates. These proteins are detectable in maternal blood and were reported to be useful as prenatal markers for pregnancy diagnosis and embryonic viability in various domestic and wild ruminants.
(Sasser et al.,1986; Zoli et al.,1992;Mialon et al., 1993;Perenyi et al., 2002; Szenci et al., 2003; Ayad et al., 2007, Panasiewicz et al., 2007)
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Conti Conti

MW ranges- 37-78 kDa.

(Butler et al.,1982; Sasser et al., 1989; Zoli et al., 1991)

Moreover, PAG have also roles in placentogenesis, detection of twins, sex of the foetus.
(Patel et al.,1995,1997; Vasques et al., 1995; Willard et al., 1995;Green et al., 2005; Lopez-Gatius et al., 2007)

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PAG gene
The PAG gene was first cloned in cattle & Sheep (Xie et al.,1991)

Later on in many speciesgoat (Garbayo et al.,2000), pig (Szafranska et al.,1995) & other species (Xie et al.,1997). cDNA microarray analysis demonstrated expression of several PAG molecules early as day 7 to 14 of pregnancy or even earlier in cattle (Ushizawa et al.,2004).
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PAG 1 GENE (CATTLE)

First gene to be cloned in cattle. 9 EXONS (99- 281 bp) 8 INTRONS (87-1800 bp) Gen-Bank DATA
(Xie et al .,1995)

ACC. NO
L27833 L22834 L27832 L27833

gDNA SEQ.(bp) Exons 1-6 Exon 7-8 Exon 9 1260 (Promoter)

IDENTIFIED BY Xie et al., 1995

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PAG 1 GENE PROFILE IN VARIOUS SPECIES

SPECIES CATTLE SHEEP GOAT PIG

IDENTIFIED BY Xie et al.,1991 -doGarbayo et al.,2000 Panasiewicz, 2004

mRNA(bp) 1295 1266 1143 1353

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Conti
Recombinant protein technology has provided
avenue for the generation of sufficient amount of specific proteins of interest from a single gene,

used for the development of improved diagnostics for early pregnancy diagnosis & conceptus viability. (Patel et al.,2004)

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PAGs expression profile

CELLS BINUCLEATE CELLS PAG profile 1,3,4,5,6,7,9

MONO- & BINUCLEATE CELLS Days

7 7 to 14 14 to 21

2,8,10,13 PAG profile

4,5,6 11,16,17 1,5-7,9-13,1517,19,21
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(Green et al.,2000; Ushizawa et al.,2004)

PAG 1
Molecular weight - 67 kDa Various isoforms (PI:4.4, 4.6, 5.2 & 5.4) Amino acids -380

Bovine and ovine PAG1 shows 73% aa identity

Proteolytically inactive

Belong to boPAG1 group

(Xie et al.,1991; Zoli et al.,1991;Guruprasad et al.,1996; Sousa et al.,2006)
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Conti...
Half-life is approximately 7-9 days.
(Kirakofe et al.,1993)

Becomes detectable soon. After implantation is initiated. Detected by RIA and ELISA
(Zoli et al.,1992; Perenyi et

al.,2002; Green et al.,2005)

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PAG 1 PROTEIN PROFILE IN VARIOUS SPECIES

SPECIES CATTLE SHEEP GOAT HORSE ZEBRA MOL.WT (kDa) 37-78 47-90 55,59,62 37,41 AMINO ACIDS 380 382 341 388 388 pH 4.0-4.4 4.0-6.8 4.9-6.2 4.5 -

DEER
PIG

30,50,55
43

380
389

5.6-6.2

(Szafranska et al.,2006)
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DIAGNOSTICS DEVELOPED USING PAG 1 PROTEIN (CATTLE)

RIA 497 (PURIFIED) Diagnose pregnancy & non-pregnant -

(Zoli et al.,1992) 93% 97%

Overall accuracy
RIA 706 & 708 (SP) ELISA SYSTEM (SP)

94%
(Perenyi et al.,2002) (Green et al.,2005)

KIT DEVELOPED FROM PAG 1- BioPRYN

(Sasser,2006)

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PAG1 concentration (ng/ml) in blood of cattle

Able to detect PAG molecules in the maternal blood as early as 21 days post AI Compared three established RIA (Perenyi et al.,2002)

Days after AI 21 32 42

RIA I 0.43 2.43 4.01

RIA II 0.48 4.17 5.60

RIA III 0.64 4.30 5.56

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PAG 1 PROTEIN PROFILE IN VARIOUS SPECIES

SPECIES CATTLE SHEEP GOAT HORSE ZEBRA MOL.WT (kDa) 37-78 47-90 55,59,62 37,41 AMINO ACIDS 380 382 341 388 388 pH 4.0-4.4 4.0-6.8 4.9-6.2 4.5 -

DEER
PIG

30,50,55
43

380
389

5.6-6.2

(Szafranska et al.,2006)

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THANK YOU

(c) Jerome A